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Coupling an universal primer to SBE combined spectral codification strategy for single nucleotide polymorphism

Mílton Cordeiro¹, Letícia Giestas, João C Lima

¹CIGMH, Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Campus de Caparica, 2829-516 Caparica, Portugal; REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Campus de Caparica, 2829-516 Caparica, Portugal.

Journal of Biotechnology

|August 15, 2013

Summary

This study presents a universal method for single nucleotide sequence discrimination using Förster resonance energy transfer (FRET) and single base extension (SBE). The approach accurately detects allele variants in solution, enhancing genetic analysis capabilities.

Keywords:

FRET SBE SNP Spectral codification Universal SBE primer

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Area of Science:

Molecular Biology
Biotechnology
Genetics

Background:

Previous work established a Förster resonance energy transfer (FRET) based spectral codification combined with single base extension (SBE) for nucleotide sequence discrimination.
This method demonstrated unequivocal detection of allele variants in solution.

Purpose of the Study:

To develop a universal approach for single nucleotide sequence discrimination applicable to any locus of interest.
To combine sequence-specific primers with universally labeled oligonucleotides for enhanced spectral codification.

Main Methods:

Development of a universal strategy employing a labeled oligonucleotide primer (donor).
Utilizing a sequence-specific primer for the incorporation of an acceptor-labeled dideoxynucleotide triphosphate (ddNTP).
Discrimination of allele variants through Förster resonance energy transfer (FRET) signals between donor and acceptor pairs.

Main Results:

Successful extension of the FRET-SBE strategy to a general, locus-independent method.
Demonstrated unambiguous allele variant discrimination via distinct FRET signatures.
Validated the incorporation of acceptor-labeled ddNTPs directed by sequence-specific primers.

Conclusions:

The developed universal strategy enables broad application of FRET-SBE for genetic analysis.
This method provides a robust platform for single nucleotide polymorphism (SNP) genotyping.
The approach offers high specificity and sensitivity for allele variant detection in solution.